| Coagulation Factor XIII (FXIII) is a transglutaminase that strengthens a blood clot by catalyzing the formation of isopeptide bonds between the side chains of specific lysine and glutamine residues of noncovalently associated fibrin chains. In vivo, the enzyme is activated through proteolysis by thrombin in the presence of calcium. Alternatively, FXIII may be activated nonproteolytically through exposure to high concentrations of calcium or sodium. Hydrogen/deuterium exchange (HDX) followed by MALDI-TOF mass spectrometry was applied to the various activated states of Factor XIII. Our results revealed subtle changes in solvent accessibility localized to regions of the beta-sandwich, activation peptide, beta-barrel 1, and catalytic core domains relative to zymogen Factor XIII. One area of the enzyme experiencing increased solvent accessibility upon activation, spanning residues 220--230, has been previously implicated as a substrate recognition region. Key changes in solvent accessibility occurring upon activation of Factor XIII were strongly dependent on the presence of calcium. Factor XIII was activated equally well by high concentrations of sodium chloride or choline chloride in the absence of thrombin, which suggested the ionic strength environment of Factor XIII plays a significant role in its activation. Specific labeling of cysteine and lysine residues using alkyl maleimides and acetic anhydride, respectively, detected changes in solvent exposure of several key residues upon activation of Factor XIII. These include the active site Cys 314, Cys 409 located near the dimer interface of the a2 dimer, Lys 221, and beta-barrel 2 residues Lys 677, Lys 678, and Cys 695. Lys 221 is located within the peptide 220--230; increased solvent exposure of this residue upon activation confirmed our HDX results. The proximity of Cys 409 to the non-prolyl cis peptide bond Q425-F426 is interesting, and increased solvent exposure of this residue may be related to the proposed conversion of Q425-F426 to the trans conformation upon activation of Factor XIII. A series of surface plasmon resonance experiments were performed to investigate the binding interactions among FXIII, thrombin, and fibrinogen. Our results indicated a system in which thrombin binds to fibrinogen faster than to Factor XIII, and dissociates from fibrinogen more slowly than from Factor XIII. |
